PKR is a key component of the cellular innate immune response against viral or bacterial infection and is an important mediator and integrator of signals that control other diverse cellular processes such as the cell cycle, cell growth, apoptosis, and the response to cell stresses such as growth factor deprivation. Aberrant PKR function is also associated with human diseases including cancers, neurodegenerative diseases and, potentially, metabolic disorders and obesity. Our understanding of PKR regulation by RNA, or other molecules, and its potential as a drug target are currently hampered by the lack of high-resolution structural information for a PKR-RNA complex. This proposal describes a research program to obtain crystals of a double-stranded RNA-activated protein kinase (PKR):non-coding viral RNA complex in order to produce the first high-resolution molecular snapshot of this important cellular kinase and its regulation by RNA. Our approaches to this major challenge are grouped into two integrated aims. First, since the most critical determinant of success in RNA crystallography is the RNA construct itself, Specific Aim 1 will exploit the detailed biochemical understanding we have developed of the non-coding adenoviral transcript VA RNAI to produce a diverse 'library' of novel RNA constructs for crystallization with PKR. These include systematic variation of the RNA structural domains, introduction of compact and stable RNA secondary structures such as tetraloops and tetraloop receptors, and incorporation of specific binding sites for other proteins (U1A or a recently developed RNA Fab) that can promote crystallization. We have also developed a rigorous strategy that will be used to ensure that each of these novel VA RNAI constructs retains wild-type PKR-inhibition activity, including global RNA folding analysis by UV melting, PKR autophosphorylation inhibition functional assays, and qualitative or quantitative measurement of PKR-RNA binding by gel filtration chromatography or isothermal titration calorimetry, respectively. Specific Aim 2 will employ high-throughput automated approaches to crystallization and X-ray diffraction screening to produce the necessary crystals. Initial experiments in this aim will be iterative in that first successes in crystallization or low resolution structure determination will feed back into the approaches of Aim 1 to refine the RNA construct design and ultimately produce crystals suitable for high-resolution X-ray crystal structure determination. The structure of the PKR-RNA complex will be determined by molecular replacement using the PKR kinase domain structure, other protein (U1A or Fab), and/or RNA fragments as the starting model(s). Alternatively, selenomethionine incorporation into protein components of the complex will be used to obtain experimental phases to determine the structure. These studies will thus provide a critical structural breakthrough that is a prerequisit for mechanistic studies of PKR and the future development of this important enzyme as a therapeutic target via structure aided design of small molecule inhibitors.